To clarify the oxygen transport across the microvessel and oxygen distribution in the skeletal muscle, we have newly designed an intravital laser microscope with oxygen-dependent quenching of phosphorescence technique. The phosphorescence probe for oxygen was injected into the systemic blood, and phosphorescence excited by N_2/dye pulse laserin the tissue area of 15 mum in diameter was measured by a photomultiplier. In vitro and in vivo oxygen tension (pO2) measurements revealed the efficiency of this method because of its non-invasiveness, accuracy, and rapid response time. This technique was applied to pO2 measurements in the rat microcirculation. The arteriolar and venular pO2 measurements were performed at the several regions classified by branching order. The mean arteriolar pO2 of different orders decreased according to increase the branching(A1 : 72.4, A2 : 51.8 and A3 : 44.1 mmHg), however venular pO2 were independent on vessel orders(V1 : 27.5, V2 : 28.6 and V3 : 32.0 mmHg). Interstitial pO2 adjacent to arterioles of different orders were significantly lower than those of intravascular pO2 (I1 : 47.8, I2 : 35.8 and I3 : 24.4 mmHg). In conclusion our new designed intravital microscope was proved the effective method to clarify the mechanisms of oxygen transport across the microvessels and the oxygen distribution in the skeletal muscle. In addition, the existence of significant reduction of pO2 levels in arterioles of different orders and large pO2 gradients at the interface between blood and tissue suggest that oxygen consumption of the smooth muscle and/or endothelium in the skeletal muscle arteriole might be high.